Throughout this application various references are referred to within parenthesis. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding claims.
In order to follow the dynamics of single cellular microtubules in living cells using time-lapse imaging, expression at high levels of Green-Fluorescent Protein linked to the microtubule associated protein were required. Traditional Green Fluorescent Protein labeling methods induced artifacts such as micro-tubule bundling, mitotic abnormalities, photo bleaching (Heim, et al., 1996) (Cormack et al., 1996) and on/off blinking (Dickson et al, 1997). These labeling induced changes in cell morphology limits the usefulness of traditional Green Fluorescent Protein labeling in vivo.
This invention discloses a method for observing in vivo dynamics and function critical for many cellular processes in living cells using a Green Fluorescent Protein chimeric molecule (GFP-Ensconsin) to provide a non-perturbing label of cellular components.
This invention discloses that the Green Fluorescent Protein chimeric molecule produces a strong stable fluorescence signal useful in labeling individual protein components making them visible for quantitation by fluorescence speckle microscopy and time lapse imaging.
This invention provides a vector comprising an isolated nucleic acid which encodes a nucleic acid segment of interest linked to one or more nucleic acid segments encoding at least two Green Fluorescent Proteins.
This invention provides a method for detecting a polypeptide of interest in a living cell which comprises: (a) transfecting the living cell with an isolated nucleic acid which encodes the polypeptide of interest linked to at least two Green Fluorescent Proteins. Additional polypeptides different from the polypeptide of interest may also be linked to at least two molecules of Green Fluorescent Protein; (b) culturing the transfected cell in conditions permitting expression of Green Fluorescent Protein and the polypeptide of interest; and (c) detecting the fluorescence of the Green Fluorescent Protein, thereby detecting the polypeptide of interest in a cell.